Quantification of fibrin in blood thrombi formed in hemodialysis central

venous catheters: a pilot study on 43 CVCs

Thabata C. Lucas*1, Francesco Tessarolo2,3, Patrizia Veniero4, Elvira D’Amato3, Iole Caola5,

Giandomenico Nollo2,3, Rudolf Huebner1, Giuliano Brunori4

1Department of Mechanical Engineering, Universidade Federal de Minas Gerais, Antônio Carlos

Avenue 6627, 31270901 Belo Horizonte, Brazil.

2Department of Industrial Engineering & BIOtech Center for Biomedical Technologies,

University of Trento, via delle Regole 101, 38123 Mattarello Trento, Italy

3Healthcare Research and Innovation Program, Bruno Kessler Foundation, Via Sommarive 18,

38123 Povo Trento, Italy

4Division of Nephrology, S. Chiara Hospital, Largo Medaglie d’oro 7, 38123 Trento, Italy

5Department of Medicine Laboratory, Azienda Provinciale per i Servizi Sanitari di Trento, via

Degasperi 79, 38123 Trento, Italy

Running Title: Fibrin quantification in CVC thrombus

Financial support: The study is part of the research project “RuBiCat - Role of the Biofilm and

device modification for the Prevention of Catheter related infections”. The project is co-funded

by the Autonomous Province of Trento, Grant 2008, and Fondazione Cassa di Risparmio di

Trento e Rovereto, Young Researcher Grant 2009.

Conflict of interest: none to declare

*Corresponding Author:

Thabata Coaglio Lucas

Bioengineering laboratory, Department of Mechanical Engineering

Federal University of Minas Gerais

Antônio Carlos Avenue 6627, 31270901 Belo Horizonte MG (Brazil).

Tel: +55 31 34096677

Fax: +5531 98280379

Email: thabataclucas@yahoo.com.br

Abstract

Purpose: Fibrin deposition and thrombotic occlusion represents a serious cause of access

disfunction in hemodialysis central venous catheters (CVCs). The aim of this work was to define

and apply a method for imaging and quantifying fibrin in thrombi formed into the side holes of

CVCs.

Methods: Forty-three CVCs removed from a cohort of dialysed patients were analysed in this

pilot study. Eosin and hematoxylin and a modified Carstair’s staining were applied on permanent

thrombus sections. Fluorescence microscopy and image analysis were performed to quantify

fibrin amount.

Results: Highly fluorescent areas were invariably associated with fibrin by the Carstair’s method.

The deposition of concentric layers of fibrin and erythrocytes were easily identified by

fluorescence microscopy showing growth features of the thrombus.

Fibrin amount in diabetic patients was significantly higher than in non-diabetic patients with a

median (IQR) values of 51(47-68)% and 44(30-54) % respectively (p=0.032).

No significant difference in fibrin content was found by grouping data according to catheter type,

permanence time, insertion site, and dialysis vintage. Higher variability in fibrin values was

found in thrombi from CVCs removed after 1-15 days in respect to 16-60 days. A trend to an

increase of fibrin amount in thrombi was noted according to blood platelets count at CVC

insertion.

Conclusions: The analytical method here presented proved to be a rapid and effective way for

quantifying fibrin content in thrombi formed on CVCs with potential application in future

clinical studies.

Introduction

Tunnelled and non-tunnelled central venous catheters (CVCs) performs a primary role to correct

acute metabolic derangements caused by renal dysfunction, an important ancillary role during

grafts and fistulae maturation in patients with chronic kidney disease (CKD) and an additional

venous access for patients during the braking-in of peritoneal dialysis(1,2). Proper catheter

management is aimed at preserving lumens patency and minimize the risk of infection. Among

non-infective complications, vascular access failure due to thrombus formation is a common

reason for hospitalization among hemodialysed patients(2,3,4). This issue has increased the interest

in clinicians and manufacturers for ameliorating CVCs performance and lowering risk of

thrombus formation to finally provide an adequate and reliable dialysis for both short and long

term catheterization. The National Kidney Foundation guidelines from the “Dialysis Outcomes

Quality Initiative” set the lower limit to 300 ml/min for extracorporeal blood flow to properly

treat the patient without excessive duration of the procedure(3,4,5). Insufficient blood flow is a

signal of catheter malfunction, frequently related to thrombotic obstructions(1,2,4,6,7). Intraluminal

catheter thrombi occur when a blood clot partly or completely occludes lumens or catheter holes.

Different catheters are equipped with a number of side-holes of various sizes and positions on

the catheter wall for maximizing blood flow, but simultaneously inducing vortexes and shear

stresses that facilitate the thrombus formation. Fibrin deposition during the initial thrombus

formation and mature phase with lumen clogging are both important steps to characterize in

respect to thrombotic tissue morphology and composition. Fibrin is composed of fibrinogen,

albumin, lipoproteins, and coagulation factors. A fibrin sheets around the CVC is documented to

form within 24 hours from CVC insertion(3,4,6,7). The fibrin sheath attracts platelets and

coagulation factors and promotes leukocyte adherence. Further layers of fibrin are then deposited

on the CVC surface creating fibrin network and plaques able to trap large amounts of

erythrocytes. This mechanism can result in the clogging of CVC side holes and lumens. We

recently proposed scanning electron microscopy and two photons laser scanning microscopy as

effective techniques for imaging and characterizing the superficial composition of thrombi in

CVC(8). However, sub surface layers of thicker thrombi (i.e. > 100 µm in thickness) remain

hidden to this analysis. The purpose of this work was to define a method for imaging and

quantifying all fibrin layers in thrombi formed into the side holes of hemodialysis CVC removed

from patients. Specific histochemical techniques, fluorescence imaging and image analysis will

be described and applied to a set of CVC thrombi collected from an observational clinical trial.

Materials and methods

CVC collection and thrombus processing

A total number of 43 CVC (30 non-tunnelled CVC, MedCOMP/HEMO-CATH®, Harleysville,

PA, USA and 13 tunnelled CVC, Palindrome TM, Covidien, Mansfield, MA, USA) were collected

from 36 patients (patients median age (range) 63(33-83) years; male:female 23:13; 97%

Caucasians; 33% diabetic), recruited in the period from August 2011 to May 2013 at the

Nephrology Division of the Trento Hospital in Italy during a prospective observational study.

The study was approved by the Ethics Committee for Research Involving Human Subjects and

Clinical Trials (Process 04/2010) of Trento, Italy. A data collection form with essential

information about patient and device was filled by the attending nephrologist at the time of

catheter removal. Prothrombin time (PT), Partial thromboplastin time (PTT) and blood platelets

count were obtained at time of CVC insertion and removal. Catheters were prevalently placed in

jugular (56%) or subclavian (40%) vein and occasionally in the femoral vein (4%). Eighty

percent of the catheters were used to perform a three hours long hemodyalisis procedure trice a

week. Median (range) insertion time was 16(2-43) days for non-tunnelled CVC 140(26-987)

days for tunnelled CVC). CVCs were removed both for infective or non infective reasons (21%

for suspected or confirmed catheter related infection, 72% at elective removal, 7% for

malfunctioning). Dialysis vintage was also computed as the length of time in months that the

patients spend on dialysis up to the CVC insertion. Thirty-four CVCs were used to perform the

first dialysis procedure on the patient, seven CVCs were placed in patients with a dialysis vintage

ranging from 2 to 72 months.

All CVCs were rinsed in sterile saline immediately after removal from patient to reduce excess

of blood and then fixed in 4% buffered formaldehyde. A photographic documentation of the

catheter was carried out and the proximal 10 cm shaft section was separated from catheter body

with a scalpel after a minimum fixation time of 48h. The catheter segment for histological

analysis was cut in two halves by transversal sectioning through the centre of the catheter hole by

a scalpel. This procedure produced two equivalent samples available for histological processing

and characterization according to the protocols described below. Catheter tips from a

representative tunnelled and non-tunnelled CVC are reported in Fig. 1, showing the sample

processed for histology.

Histochemical staining

Two samples obtained from each catheter were processed routinely for obtaining permanent

sections. Briefly, samples were dehydrated by immersion in ascending hydro-alcoholic solutions,

100% ethanol and finally 100% xylene. After paraffin embedding, 5 µm thick sections were cut

with a rotary microtome. At this stage, CVC slices were carefully removed from each section

because of poor adherence to the glass slide leaving only the thrombus slice for the following

histochemical staining. Orientation of the thrombus section was easily identifiable by the specific

“D” shape of the thrombus portion inside the lumen.

Two consecutive sections were stained respectively with hematoxylin-eosin stain (H&E) and a

modified Carstairs’ stain for comparison. H&E stain was performed by 10 minutes in Mayer’s

hematoxylin and 30 seconds in eosin. Because platelets and fibrin within a thrombus cannot be

easily differentiated by H&E staining, we adapted the original Carstairs’ staining method (9) to

identify platelets and fibrin in the thrombus sections. Staining times and fixation solutions were

optimized for a better differentiation of thrombus components. Briefly, paraffin sections were

hydrated, placed in 5% ferric alum for 5 minutes, rinsed in running tap water, stained in Mayer’s

hematoxylin for 5 minutes, and then rinsed again in running tap water. Slides were placed for 4

minutes in picric acid-orange G solution (20 mL saturated aqueous picric acid, 80 mL saturated

picric acid in ethanol, and 0.2 g orange G) and then rinsed in distilled water. Slides were then

placed in ponceau-fuchsin solution (0.5 g acid fuchsin, 0.5 g ponceau 2R, 1 mL acetic acid, and

distilled water to 100 mL) for 2 minutes and then rinsed in distilled water. Slides were treated

with 1% phosphomolybdic acid for 3 minutes and then rinsed in distilled water. Slides were

finally stained with aniline blue solution (1 g aniline blue in 100 mL 1% acetic acid) for 30

minutes, decoloured in 1% aqueous acetic acid for 3 minutes, rinsed in several changes of

distilled water, dehydrated, cleared, and then mounted with acrylic medium. In sections obtained

from thrombi fixed for 48 hours or longer, Carstairs’ method produces differential staining of

fibrin (bright red), platelets (grey-blue to navy), collagen (bright blue), muscle (red), and red

blood cells (RBC) (yellow). Fig. 2 shows fibrin layers, platelets and erythrocytes in a

representative sample from a CVC. Carstairs’ stained slices were observed in bright field

transmitting light microscope (DMIL, Leica, Wetzlar, Germany) at a magnification of 40x, 100x,

and 200x. Corresponding H&E stained slides were observed both in transmitted white light and

under green light for evidencing red fluorescence among components of the thrombus tissue.

Fluorescence images from H&E stained sections were compared to images obtained from

Carstairs’ stained sections to identify the fluorescent thrombus components and to check

specificity of fluorescence signal.

Fluorescence microscopy and image analysis for quantification of thrombus composition

Three additional sections were obtained at different sample depth and stained with H&E stain.

H&E stained slices were used for quantifying fibrin amount by fluorescence microscopy. One

high resolution image (2592 x 1944 pixels, RGB 16 bit) per slice was obtained by using a 4x

magnification objective (Leica C PLAN, NA=0.10) The excitation light was obtained from the

green line (546 nm) of a mercury lamp and red fluorescence was collected by a CCD colour

camera (DMIL 420, Leica, Wetzlar, Germany). Acquired images were imported into Image J

software (NIH, USA) and converted to 8 bit grey-scale. Binary threshold was applied twice: a

first low grey value threshold identified the total area occupied by the thrombus section and a

second medium grey value threshold identified highly fluorescent tissue within the section as

described in Figure 3. The total number of white pixels in the two binary images accounted

respectively for the total thrombus area and the highly fluorescent area. Means and standard

deviations of the ratio between highly fluorescent and total thrombus area were eventually

computed per each sample.

Clinical data and statistical analysis

Quantitative data of fibrin amount obtained from image analysis were statistically analysed for

correlations with blood coagulation parameters (PT, PTT and blood platelets count) by Spearman

rank test. Data were also grouped according to CVC type (tunnelled or non-tunnelled)

permanence time of the catheter into the vein of the patient (0-15 days; 16-60 days; more than 60

days), diabetic patient status (yes or no), patient dialysis vintage (catheter used for starting

dialysis or inserted after more than 1 month of dialysis), catheter insertion site (jugular,

subclavian or femoral vein). Descriptive statistics of groups were expressed as median and

interquartile range (IQR). Distributions of fibrin amount value in each group were checked for

differences by Mann-Whitney or Kruskal-Wallis test for two or more than two independent

groups respectively. Statistical significance was considered for p<0.05. Statistical analysis was

performed using statistical software SPSS 16.0 (SPSS Inc., Chicago, Illinois, USA).

Results

Qualitative and quantitative histological analysis was feasible in each of the 43 samples included

in the study. Carstairs’ stained slides allowed fast preview of the main fibrin structure by low

magnification optical microscopy, showing fibrin fibres, bundles and plaques in red and packed

erythrocytes in yellow. A high variability in fibrin and erythrocytes amount was present among

samples, but a layered concentric structure made of alternated fibrin and erythrocytes layers was

invariably found in all samples. Concentric layers were generally more regular and circular in the

thrombus region close to the lumen wall, while differences were present in the thrombus area

facing the catheter hole. Figs. 4, shows sections obtained from three different samples,

representatives for three different thrombus compositions: prevalence of fibrin (Fig. 4a), fibrin-

erythrocytes mix (Fig. 4d), prevalence of erythrocytes (Fig. 4g).

The corresponding images, acquired by detecting fluorescence signal on consecutive slice

stained with H&E, are presented in Figs. 4b, 4e and 4h. The comparison with Carstairs’ stained

sections showed a good correspondence between fuchsine stained and red fluorescent areas, thus

confirming that fluorescence signal can be invariably ascribed to fibrin. Binary images in Figures

4c, 4f and 4i were obtained by applying a threshold to fluorescence images and show fibrin

distribution in white. Fibrin quantification gave a percent fibrin value (percent ratio between

highly fluorescent and total thrombus area) of 63%, 44% and 23% for figs. 4c, 4f and 4i

respectively.

Data on fibrin amount grouped according to CVC type or CVC permanence time into the vein of

the patient are summarized in Figures 5a and 5b respectively. Non tunnelled and tunnelled CVCs

showed a median (IQR) fibrin value of 45(41-55)% and 48(27-57) respectively. No significant

difference in fibrin amount between the two catheter types was found.

Groups created according to CVC permanence time accounted for 13, 21 and 9 CVCs. Median

(IQR) values for fibrin amount in each group were 44(41-72) %, 45(39-47)%, and 49(46-57)%

for a catheter permanence time of 0-15 days, 16-60 days, and more than 60 days respectively. No

significant differences between groups were found.

Groups created according to the diabetic status of the patient are reported in Figures 6a. Fibrin

amount in diabetic patients was significantly higher than in non-diabetic patients with a median

(IQR) values of 51(47-68)% and 44(30-54) % respectively (p=0.032).

No significant difference in fibrin amount was found for thrombi grouped according to patient

gender, insertion site and dialysis vintage.

Statistical analysis did not elicited any significant correlation between fibrin amount in thrombi

and PT, PTT and blood platelets count at CVC insertion or removal. However a trend to an

increase of fibrin amount in thrombi was noted in relation with blood platelets count at CVC

insertion when clustering data into three ranges (<150000, 150000-300000, >3000000 per µl ) as

reported in Figure 6b.

Discussion

The formation of an intraluminal venous thrombus in dialysis catheters is primarily related to

CVC dysfunction, but deserve attention because pulmonary embolism, infection and loss of

central venous access can follow(1,10).

Removal of blood through the CVC holes creates a negative pressure around these holes which

can cause the vein wall to collapse around the holes and obstruct flow promote fibrin formation.

If a fibrous tissue sheath forms around the catheter extending to the CVC tip or if clots form

around the tip and side holes, the entry hole to the catheter becomes smaller and the velocity of

blood flow can be increased significantly(11). Variations in blood velocity can induce

recirculation areas between arterial and venous holes, leading to turbulent flow. In the

recirculation areas, fibrin starts developing on the catheter wall and propagates primarily along

flow direction. Due to hole design and local turbulence, lateral aggregation is also promoted in

the proximity of the hole border and eventually a fibrin plaque able to definitely obstruct the

CVC hole is formed(11). Furthermore, recirculation and turbulent flow can be locally affected

also by fibrin sheaths, clots, and the relation of the catheter tip and side holes to the vascular

surfaces. The regions of disturbed flow are characterized by flow separation and flow eddies

zones with low values of shear stress and could contribute to an increased residence time of

blood components on the CVC surface. This may s result in the stimulus of platelet aggregation

by prolonged contact with the polymeric CVC. Previous studies reported that platelet

aggregation preferentially occurs at regions of low shear stress, i.e. in flow eddies and in flow

separation regions(12-14). In the flow eddies zone, the wall shear stress has been reported to

remain at a low level (<1.0 Pa) throughout the cardiac cycle due to the reversed flow

conditions(13, 14).

Also the presence of high blood flow rate into the CVC holes during the hemodialysis procedure

promotes the adhesion of a high numbers of platelets on the CVC wall. In this situation, the

adhesion is controlled by the reaction kinetics of the interaction of the platelet membrane

receptors and the proteins adsorbed on the catheter surface (15). A recent study compared the

structure of thrombi formed in the right atrium and in pulmonary arteries showing that thrombi

formed in arteries with high flow velocity has lower number of erythrocytes, accompanied by

increased fibrin content (16). The high concentration of fibrin in the thrombus can impede, in

clinical practice, the dissolution of the thrombi despite administration of anticoagulants (16).

Similarly, the high amount of fibrin which surround the thrombus can do ineffective the

anticoagulants into the lateral holes of CVC. Therefore even when anticoagulant is administered,

catheter dysfunction could happen and manifest by failure to aspirate blood from the lumen,

inadequate blood flow and/or high resistance/pressure during hemodialysis.

The interactions between blood and the CVC polymer surface begins with the adsorption of

proteins as gamma globulin, fibrinogen and von Willebrand factor that elevates platelets

adhesion and activation, thus increasing the amount of fibrin on the device(17). This process

begins minutes after the CVC insertion since the polymeric surface permit platelet adhesion and

activation of the intrinsic and extrinsic coagulation pathway (17).Protein adsorption determines

the activation and modulation of the coagulation cascade complement system, platelets and

immune cells guiding their interplay. This results in the formation of a transient provisional

matrix and the onset of the inflammatory response that occur on CVC surfaces. The synthetic

material induces fibrinogenesis and causes the construction of numerous fibrin strands which

may eventually result in a fibrous capsule around the catheter (18, 19).

The methods presented in this study, based on Carstairs’ staining and fluorescence imaging of

thrombus sections clearly identified the layers of fibrin which first developed on catheter lumen

and lateral holes surface and subsequently grew toward the centre of the lumen in a layer by

layer process. Hechler and coworkers performed Carstairs’ staining and ultrastructural analysis

to analyze the early phase of thrombus formation in jugular veins in mice models (20). Authors

reported that the initial thrombus was constituted of a central area rich in erythrocytes with

platelets in the periphery. Similarly, in this work, the histological examinations of thrombus

sections stained by Carstairs’ evidenced erythrocytes accumulated mainly in central area while

fibrin and platelets accumulates in the peripheral regions of the thrombus. This arrangement is

evident in Fig.2. The initial fibrin fibres are generally aligned parallel to the tangent of the hole

wall of the catheter; as the thrombus grows the layers of fibrin infiltrates and surrounds the

tissue. The deposition of concentric layers of fibrin increases with the development of the

thrombus as can be seen in figs. 4e and 4b. In addition, the circumferential direction of the fibrin

suggests the influence of the substrate in fibrin alignment. A similar arrangement was found in

vascular clots within the venous vessels(21). Fibrin has an anisotropic molecular structure, and the

orientation of the fibrin fibre bundle could be quantitatively assessed by polarized light

microscopy(21). In addition the combination of picrosirius red staining and polarized light

microscopy provided a method of identifying fibrin and differentiating it from other blood

elements(21). However, to obtain a comprehensive visualization of the fibrin fibres, circularly

polarized light has to be used. Whittaker and Przyklenk proposed a detailed description for the

imaging apparatus(21), but it is not widely diffused in clinical setting. Conversely, linear

polarizers are usually available at the histology facilities, but allow the visualization of fibres

alligned to the polarization axis, showing only a part of the total amount of fibrin in the tissue

section. In this study, the proposed method for fibrin quantification relies on conventional

histochemical staining and fluorescence microscopy. The methodology can be easily

implemented in histology departments for a rapid differentiation and quantification of fibrin

among other blood elements in the thrombus.

By combining Cartairs’ staining and fluorescence microscopy we were able to define a method

to quantify the fibrin amount of the thrombus in hemodialysis CVC. The content of fibrin in

thrombi is generally reported to increase with thrombus maturation. Results from this study

confirmed this trend. Interestingly, the analysis of thrombi formed on non-tunnelled catheters

showed a high variability for data coming from catheters with a permanence time between 1 and

15 days. Differently, non tunnelled CVCs inserted for 16 to 60 days presented a low variability

in fibrin amount. This can be partly motivated by considering that in the initial thrombus

formation there is a more variable fibrin architecture that becomes more structured in older

thrombi. Fibrin fibres alignment was also seen to increase with the time in the mouse model (22).

Mature thrombi have therefore a coarse fibrin network with a majority of thick fibres, while thin

and thick fibrin fibres are present in young thrombi. This can contributes to the variability of the

fibrin-covered area in the catheters with 0-15 days of CVC permanence. From the clinical point

of view, patients interested by short time catheterization (1-15 days) are usually critical patients,

showing a higher inflammatory status or even sepsis that can induce altered reaction to foreign

materials put in contact with tissues. Above these aspects, variation in fibrin amount is related

also to size and conformation of the thrombus, number of hemodialytic procedures, intensity of

aphaeresis flux, incidence of previous lumen occlusions and successful urokynase procedures,

and specific haematic parameters of the patient. Among the coagulation parameters that affect

the coagulation pathway, prothrombin consumption, thrombin activity, fibrinogen level and

platelet count are deeply involved in the variation of fibrin amount in thrombi. Increased

thrombin activity on platelet surfaces has directly been attributed to fibrin mesh architecture (21).

The concentration of thrombin deeply affects fibrin architecture (18) and thrombus structure,

varying the cross-linking process among fibres that join together into aligned band patterns (23,

24). Branch points make fibres a stable network, providing strength and stability to the thrombus

tissue. Considering these issues we collected values of PT, PTT and blood platelet count of the

catheterized patients included in this study. Although no significant correlation was found

between these three parameters and the amount of fibrin in CVC thrombi, fibrin content in the

thrombus tended to increase according to the increase of the blood platelet count of the patient.

Larger studied studies should include the monitoring of these parameters and other coagulation

factors.

Besides possible correlations between blood parameters and thrombus composition, results of

this study elicited a significant difference in fibrin amount according to the diabetic status of the

patient. The higher amount of fibrin in CVC thrombi from diabetic patients was never reported

before, but diabetes mellitus was already considered to contribute to a prothrombotic state,

including endothelial dysfunction, coagulative activation and platelet hyper-reactivity(25).

Platelets in diabetic patients are characterized by deregulation of several signaling pathways

leading to enhanced adhesion, activation and aggregation(25,26). Data on CVC thrombi

composition support these aspects and should be considered in the management of diabetic

patients needing hemodialysis.

Conclusions

A new method for addressing fibrin distribution and amount in thrombi formed on hemodialysis

CVC was reported and tested on a group of thrombi from 43 CVCs. Although different

methodologies for assessing thrombus composition have been previously reported(8, 9, 12, 21, 22), the

method here presented allowed assessing thrombus structure, fibrin content and arrangement in a

quantitative way. Results of the pilot clinical study proved evidence that fluorescence

microscopy and image analysis is an effective method to assess thrombus structure in a rapid

way with commonly available instrumentation and without any discomfort for the patient.

Although this clinical pilot study elicited some interesting trends in fibrin amount and fibrin

fibres organization within CVC thrombi, a larger clinical study should be set up to validate these

preliminary data. The method has the potential for giving additional information on thrombus

formation mechanisms, performance of hemodialysis catheters design, action of anti-thrombotic

coatings, effectiveness of anticoagulant therapies and locking solutions in properly designed

clinical studies.

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FIGURE CAPTIONS

Figure 1: Non-tunneled (a) and tunnelled (c) representative catheter with thrombi occluding the

proximal venous side holes. Dashed lines and dotted lines indicate respectively the transversal

scalpel cuts for separating the catheter shaft and for realizing the two samples halves. Samples

obtained after transversal sectioning of the same catheters are presented in (b) (non-tunnelled

CVC section) and (d) (tunnelled CVC section)

Figure 2: Transversal section of a thrombus formed into the side hole of a non tunneled CVC and

occluding the whole lumen section. Fibrin layers (bright red) and RBCs (yellow) are well

differentiated at both low (a) and higher magnification (b and c). Platelets appears in gray-blue to

navy at high magnifications and are often correlated to fibrin fibers (c). Carstairs’ stain: (a), x40,

(b) x100; c) x200).

Figure 3: Images from a representative thrombus section subjected to quantitative image

analysis. a) H&E stained section in white transmitting light. b) Red fluorescence image obtained

from illuminating with green light the same H&E stained section. c) Binary image obtained by

thresholding the fluorescence image at a low grey value. White area corresponds to the whole

thrombus section. d) Binary image obtained by threshold the fluorescence image at a high grey

value. White area corresponds to the higly fluorescent part of the thrombus section,

corresponding to fibrin. Original magnification: 40x.

Figure 4: Composition of three different thrombi representative for a prevalence of fibrin (a, b,

c), fibrin-erythrocytes mix (d, e, f), and prevalence of erythrocytes (g, h, i). Carstair staining (left

column) shows fibrin in purple red and erythrocytes in yellow. Fluorescence microscopy on

corresponding consecutive section stained with H&E and (middle column) present highly

fluorescent areas for fibrin. Binary images after image processing for fibrin quantification in

white (right column). Original magnification 40x.

Figure 5: Fibrin amount obtained by image analysis grouped according to CVC type (left) and to

CVC permanence time of the CVC into the vein (right) No significant difference between groups

was present.

Figure 6: Fibrin amount obtained by image analysis grouped according to diabetic status of the

patient (left) and blood platelets count (right). Fibrin amount in thrombi differed significantly

according to patient diabetic status (p=0.032). A trend to an increase of fibrin amount in relation

with blood platelets count at CVC insertion is visible.